Alcoholic liver disease (ALD) is a major source of alcohol-related morbidity and mortality. It is estimated that 10-15 percent of alcoholics develop fibrosis, cirrhosis and hepatocellular carcinoma. Inflammatory responses are critical contributors to disease progression. One important pathway to increased inflammation results from increased intestinal permeability and changes in bacterial microflora, resulting in activation of Toll-like receptor 4 (TLR4) on Kupffer cells. However, ethanol also directly injures hepatocytes; our preliminary data indicate that signals from injured hepatocytes are also critical to the activation of hepatic macrophages. The overall objective of this proposal is to investigate how TLR-dependent inflammatory responses, in combination with alcohol-induced hepatocellular injury, coordinately lead to pathogenesis of ALD. TLRs transduce their signals through the adaptor molecule MyD88 and members of the IL-1R-associated kinase (IRAK) family, including IRAK1, IRAK2, IRAKM and IRAK4. IRAK4, the upstream kinase, directly interacts with MyD88. While MyD88- IRAK4-IRAK1 mediates TAK1-dependent NF?B activation, MyD88-IRAK4-IRAK2 is critical for post- transcriptional control. While IRAKM interacts with MyD88-IRAK4 to mediate a MEKK3-dependent second wave NF?B activation, it interacts with IRAK2 to inhibit translation of cytokines and chemokines. Thus, IRAKM exerts an overall inhibitory effect on inflammatory responses. Surprisingly, we found IRAKM-deficient mice were protected from ethanol-induced liver injury. Array analysis led us to be interested in one particular TLR4-induced IRAKM-dependent gene, Mincle, a C-type lectin receptor, that senses non-homeostatic cell death, thereby inducing pro-inflammatory cytokine and chemokine production. Mincle expression was induced in liver of ALD patients and in mice after ethanol feeding in an IRAKM-dependent pathway. Further, Mincle-deficient mice were protected from ethanol-induced liver injury. Since Mincle is a sensor for cell death and its expression is IRAKM-dependent, we hypothesize that TLR-induced IRAKM-dependent Mincle up-regulation in Kupffer cells provides a critical link between alcohol-induced cell death and onset of inflammatory responses, contributing to the pathogenesis of ALD. To test this hypothesis, we propose the following Aims: Aim 1 Investigate the mechanism for TLR-mediated IRAKM-dependent pathway in ALD: we will investigate (1) The molecular mechanism for the TLR-MyD88-IRAKM-MEKK3-dependent pathway induced by low dose of TLR ligands; (2) The mechanism for the pathogenic role of the TLR-MyD88-IRAKM-MEKK3-dependent pathway in ALD. Aim 2 Investigate the mechanism by which Mincle-mediated signaling contributes to ALD: We will investigate (1) The molecular mechanism for Mincle-mediated inflammasome activation; (2) The cellular mechanism for the pathogenic role of the Mincle-dependent pathway in ALD. These combined molecular/cellular and pre-clinical studies will identify critical elements in the role of the TLR-IRAK-M-Mincle signaling in ALD, leading to improved therapeutics for prevention and treatment of ALD.

Public Health Relevance

Alcohol abuse is a leading cause of morbidity and mortality worldwide and recent data indicate that alcoholic liver disease affects over 10 million Americans. The long-term goals of this research project are to investigate the mechanisms by which ethanol exacerbates inflammation in the liver. Previous work has identified inflammatory responses as critical contributors to disease progression. Increased intestinal permeability and changes in bacterial microflora result in elevated levels of bacterial products in alcoholic patiens and animal models of ALD. Lipopolysaccharide (LPS) recognition by Toll-like receptor 4 (TLR4) on Kupffer cells, the resident hepatic macrophages, releases inflammatory cytokines that impact the functions of hepatocytes and stellate cells. At the same time, alcohol exposure contributes to hepatocellular injury and death. Preliminary data indicate that signals from these injured hepatocytes are also critical to the activation of hepatic macrophages. Thus, the overall objective of this proposal is to investigate how TLR-dependent inflammatory responses, in combination with alcohol-induced hepatocellular injury, coordinately lead to the pathogenesis of ALD. Understanding the complex interaction between alcohol-induced hepatocellular injury and inflammatory responses will provide the foundation for the future development of rationally designed therapeutic interventions to slow and/or reverse alcoholic liver disease.